Immunization against infection caused by pathogenic microorganism (bacteria, viruses and parasites) is generally achieved by inoculating an individual with the natural antigen (attenuated or killed microorganism) or parts of said infectious agent (for example detoxified microbial products) in order to stimulate a protective immune response able to neutralize the pathogenic microbe or its deleterious effects.
Limited availability of the natural antigenic substance, risks involved in handling pathogenic material as well as storage problems stimulated the interest in the development of subunit vaccines. Isolated protective epitopes nevertheless are often characterized by their poor immunogenicity. The carbohydrate capsules of bacteria are an example of such coats: They are not easily recognized by T cells and therefore the immune response to these antigens is deprived of T cell help, T cell memory, IgG class switch, and affinity maturation. Such an immune response is inefficient and resistance to infection with bacteria encoated with carbohydrate capsules is not easily obtained by vaccination, with bacterial carbohydrates. Peptide epitopes too may be poorly immunogenic, the absence of a T cell epitope and the genetically restricted immune response being the reason.
It is now well established that most antigens require T cell help to induce B cells to produce antibodies. Conjugating a "helper" or T cell determinant to a B cell-specific antigen was shown to induce humoral immune responses to the coupled B cell epitope. The discovery by Avery & Goebel (1929) that coupling of polysaccharides to protein carriers increases immunogenicity has recently been used for the preparation of vaccines for human use. Both in humans and in rodents these conjugates behave like T cell dependent antigens by exhibition of immunological memory. There are similarities between conjugate polysaccharide vaccines and protein carrier-hapten systems. Thus the capsular polysaccharide (CPS) conjugates are able to induce protective levels of CPS antibodies in infants, while CPS alone is not. It is possible that the superior immunogenicity of conjugates compared to that of pure polysaccharides is due to the help by carrier-specific T cells, as has been demonstrated in the carrier-hapten system in rodents.
In most cases, T cell independent (T-ind) antigens have been coupled to large immunogenic carrier proteins such as tetanus toxoid, cholera toxin or diphtheria toxoid. Nevertheless, besides dosage limitations and the risk of sensitization to the carrier itself, as reported for tetanus toxoid, the immunological response to high molecular weight carrier molecules harboring stimulatory as well as suppressive T cell epitopes is not very predictable. It has been shown that the antibody response to a hapten coupled to a carrier protein can also be inhibited when the recipient has been previously immunized with the unmodified protein. This phenomenon has been termed carrier-induced epitope suppression and was recently demonstrated to occur with a number of hapten-protein conjugates (Herzenberg & Tokuhisa, 1982). Since the development of more potent conjugate vaccines against a large number of extremely infectious organisms is still important, efforts are being made to search for more appropriate carrier molecules providing the needed T cell epitopes. Universally immunogenic T cell epitopes, defined by specific peptides with sharply outlined immunological characteristics, might represent a new generation of such alternative molecules. T cell epitopes of various sorts have been used for this purpose before. However, to trigger a strong memory response when the host meets the infectious agent after vaccination, the T cell carrier epitope should be present along with the specific B cell epitope. This fact would seem to require that a different T cell carrier be used for each infectious agent. Highly abundant proteins well recognized by the immune system might be an appropriate source for peptides serving this purpose.
Studies using a wide variety of proteins, both those closely related to self and those phylogenetically distantly related, have shown that the majority of T cells are focused onto a few immunodominant epitopes with a minority responding to other, subdominant determinants. This hierarchy of determinant utilization by T cells could result from a combination of factors including differential affinities for the available MHC molecules, the diversity of the T cell repertoire, internal competition for MHC-binding sites and fine differences in processing (Babitt et al, 1985; Kappler et al, 1987; Brett et al, 1988)
Evidence is accumulating that proteins belonging to the family of heat shock proteins (hsp's) are major antigens of many pathogens (Young et al, 1988). Hsp's were first described and later named due to their production by cells exposed to sudden elevations in temperature. The hsp's include proteins of various molecular weights,. including 20 kD, 60 kD, 65-68 kD, 70 kD, 90 kD, 110 kD, and others. It is now apparent that hsp's are induced in all cells by many different environmental insults, including oxidative injury, nutrient depletion and infection with intracellular pathogens; the hsp response enables the cell to survive under otherwise unfavorable conditions. Although cellular stress increases the synthesis of hsp's, many hsp's are also constitutively expressed and play an essential role in normal cell function. The hsp response is ubiquitous throughout the pro- and eukaryotic kingdoms and hsp's belong to some of the most conserved molecules.
Hsp65, as a representative member of the proteins belonging to the hsp family, can be considered to be a dominant antigen because infection or immunization with many different bacteria induces antibodies and T cells specific for the hsp65 molecule (Young et al, 1988). In mice immunized with Mycobacterium tuberculosis, 20% of all T cells which respond to the bacterium, are specific for hsp65. Interestingly, T cells with reactivity to hsp65 have also been identified in normal healthy individuals lacking any clinical signs of disease (Munk et al, 1988).
Lussow et al. (1990) showed that priming of mice with live Mycobacterium tuberculosis var.bovis (BCG) and immunization with the repetitive malaria synthetic peptide (NANP).sub.40, conjugated to purified protein derivative (PPD), led to the induction of high and long-lasting titers of anti-peptide IgG antibodies. Later on, Lussow et al. (1991) reported that the mycobacterial hsp65 as well as the hsp65 of E. coli (GroEL) acted as carrier molecules in mice, previously primed with BCG, for the induction of high and long-lasting titers of IgG against the repetitive malaria synthetic peptide (NANP).sub.40. Anti-peptide antibodies were induced when the malaria peptide, conjugated to the mycobacterial or E. coli hsp, was given in the absence of any adjuvants.
Barrios et al. (1992) have shown that mice immunized with peptides or oligosaccharides conjugated to the 70 kD hsp produced high titers of IgG antibodies in the absence of any previous priming with BCG. The anti-peptide antibody response persisted for at least 1 year. This adjuvant-free carrier effect of the 70 kD hsp was T cell dependent, since no anti-peptide nor anti70 kD IgG antibodies were induced in athymic nu/nu mice. Previous immunization of mice with the 65 kD or 70 kD hsp did not have any negative effect on the induction of anti-peptide IgG antibodies after immunization with hsp-peptide conjugates in the absence of adjuvants. Furthermore, preimmunization with the 65 kD hsp could substitute for BCG in providing an effective priming for the induction of anti-(NANP).sub.40 antibodies. The carrier effect of mycobacterial hsp65 and hsp70 for conjugated peptides was demonstrated also in non-human primates (Perraut et al, 1993).
It can be assumed that some T cell epitopes within the sequence of the bacterial hsp65 protein show immunodominance and are able to induce immunological memory, whereas others do not express privileged immunological recognition or are involved in the induction of autoimmune diseases. Distinguishing between functionally different T cell epitopes, binding to several different MHC molecules, may lead to the identification of universally immunogenic peptides, which can qualify as safe, defined, and potent alternatives for carrier molecules of T-ind antigens.
Israel Patent Application No. 102687 of the same applicants describes specific T cell epitopes of human hsp65, and analogs thereof, conjugated to poorly immunogenic molecules.
None of the above mentioned references describes specific T cell epitopes derived from the sequence of hsp65 of E. coli (GroEL) conjugated to poorly immunogenic molecules.